Research On Relations Between NK Cell And KIR2DL3 Gene About Inhibition Of HCV Activity In Vitro By Silencing KIR2DL3 Of NK Cells Mediated On ShRNA

Objective:This experiment aims at silencing NK cell KIR2DL3 gene by iRNA technology and research relations of NK cell and KIR2DL3 gene on inhibition of HCV activity in vitro.Then we discussed its action mechanism so as to provide a new way of thinking for HCV infection mechanism and Immune therapy.Methods:We designed shRNA approach for mRNA of KIR2DL3 gene, and Synthetized double stranded shRNA by gradient cooling.We inserted the shRNA fragments in the carrier by complementary combination through the sticy end on both sides of shRNA and inear PLKO.1 vector, then transfected expression vector with PSPAX2, PMD2G helper plasmid into 293T cells by liposome transfection method.we got lentivirus, and Incubated it with NK cells for gene expression silence of KIR2DL3 of NK cells.we examined NK cells expression of KIR2DL3 gene by western-blot.We transcripte HCV RNA from pFL-J6/JFH-1 and T7 kit, then purified HCV-RNA and transfected HCV-RNA into Hhu7.5.1 by liposome transfection methods.we collect HCV virus particles, then we separated CD4+T cells from healthy individuals by magnetic bead separation technology.At last we stablished HCV cultivation method in vitro.We tested HCV in CD4+T cells by RT-PCR. We extract NK cells and CD4+T cells from 6 subjects whose HCV antigen and antibody are negative,then we silenced their KIR2DL3 gene and infected their CD4+T cells by HCV. We incubated KIR2DL3-silencing NK cells with autologous CD4+T cells infected by HCV (HCV-CD4+T cells) for 24 hours.At last we detected The relative content of HCV-RNA in HCV-CD4+T cells by RT-qPCR.Results:We can successfully pack up stable infectious lentivirus for KIR2DL3 gene. Expression levels of KIR2DL3 protein decreased obviously in NK cells that infected lentivirus. efficiency of silencing is about 80%.We can successfully constructed HCV culture system in vitro by Liposome transfection method and Huh7.51 cells.It can generate stable HCV virus particles and infect CD4+T cells of humans successfully,The result showed that relative content of HCV was lower in the KIR2DL3-silencing group than non-silent KIR2DL3 group.Conclusion:We can successfully silence KIR2DL3 gene of NK cells by shRNA. Comparing the Killing efficiency of KIR2DL3 gene-slilencing NK cells and non-slilencing NK against HCV-CD4+T cells.We can jude preliminary that KIR2DL3-silencing Nk cells possess higher Killing activity against HCV.